Within the last thirty years, appreciation for mitochondrial genetics has expanded greatly because of discoveries demonstrating mitochondrial DNA (mtDNA) mutagenesis as causative for many pathologies affecting skeletal muscle, cardiac muscle, and the central and peripheral nervous systems. mtDNA mutagenesis has also been implicated in the process and phenotypic manifestations of aging. A major barrier to research concerning these topics and more basic ones concerning the transcriptional mechanisms of mitochondrial genetics has been an inability to develop a methodology for introducing DNA into mitochondria (i.e. transfecting mitochondria). To this end, the proposed project includes two central aims: to develop a methodology for transfection of mitochondria within cells;and to use mitochondrial transfection and genetic engineering to better understand the post-transcriptional processing of mitochondrial mRNA (mt-mRNA). To accomplish the first, two mtDNA plasmids have been developed that contain an EGFP locus. Using the fluorescence of EGFP as a marker for the presence of this mtDNA plasmid in mitochondria, we will screen established transfection protocols to observe if mitochondria have been transfected. To investigate post-transcriptional processing of mt-mRNA, the mtDNA plasmids will be used to engineer several clones to test what portions of mt-mRNA are important for processing. These studies will be carried out using an established protocol for transfection of isolated mitochondria. With better understanding of post-transcriptional processing of mt-mRNA and the ability to introduce DNA into mitochondria, researchers will be able to manipulate mtDNA, reintroduce it into a physiological system, and measure the phenotypic consequences of these manipulations. Development of transfection technology for mammalian mitochondria would yield two vital advances. First, by enabling scientists to modify the mitochondrial genome at will, and to analyze its functional properties, mitochondrial transfection would facilitate a systematic dissection of mitochondrial molecular genetics. Second, mitochondrial transfection would enable the development of cell and animal models for study of mitochondrial diseases. With the ability to create models of mitochondrial pathologies, therapeutics research would receive a significant boost, and thus, the search for treatments of mitochondrial diseases would be greatly accelerated.